Akt inhibitor-IV in estrogen receptor-positive breast cancer cells: cytotoxic, apoptotic, transcriptional, and in-silico comparison with tamoxifen

Abstract

Dysregulation of the phosphoinositide 3-kinase/protein kinase B (AKT)/mechanistic target of rapamycin pathway is a hallmark of breast cancer, making AKT a focus of therapeutic interest. AKT inhibitor-IV is a benzimidazolium compound that inhibits AKT phosphorylation, but its activity and binding mechanism in breast cancer cells have not been characterized in detail. We compared AKT inhibitor-IV with tamoxifen in MCF-7 cells. Cell viability after 24-h treatment was measured by 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide assay (AKT inhibitor-IV: 0.1–5 μM; tamoxifen: 5–50 μM); apoptotic death was quantified by nucleosome ELISA; and AKT1, BCL-2, BAX, and CASP3 mRNA levels were measured by quantitative real-time PCR. Both compounds were docked into the ATP-binding pockets of 3-phosphoinositide-dependent protein kinase-1 (PDK1) (PDB 1H1W) and AKT1 (PDB 3MVH) using AutoDock Vina v1.2.6. AKT inhibitor-IV reduced viability with an IC50 of 1.1 μM, compared with 5.2 μM for tamoxifen, and induced 5.9-fold apoptosis enrichment versus 3.65-fold for tamoxifen. AKT inhibitor-IV downregulated AKT1 (0.32-fold) and BCL-2 (0.55-fold) and upregulated BAX (5.97-fold) and CASP3 (6.43-fold) mRNA; tamoxifen showed qualitatively similar but quantitatively weaker transcriptional changes. Docking returned affinities of −9.91 kcal/mol for PDK1 and −9.66 kcal/mol for AKT1, with AKT inhibitor-IV contacting catalytic-core residues of both kinases. AKT inhibitor-IV was more potent than tamoxifen in this in-vitro setting and warrants further preclinical evaluation. Because MCF-7 cells carry the CASP3 exon 3 deletion and lack functional caspase-3 protein, the observed CASP3 mRNA upregulation reflects transcriptional reprogramming rather than restored executioner activity. The modest docking difference requires biochemical confirmation before any PDK1-versus-AKT1 selectivity claim can be established.